microorganisms Communication Metagenomic Snapshots of Viral Components in Guinean Bats Roberto J. Hermida Lorenzo 1,†,Dániel Cadar 2,† , Fara Raymond Koundouno 3, Javier Juste 4,5 , Alexandra Bialonski 2, Heike Baum 2, Juan Luis García-Mudarra 4, Henry Hakamaki 2, András Bencsik 2, Emily V. Nelson 2, Miles W. Carroll 6,7, N’Faly Magassouba 3, Stephan Günther 2,8, Jonas Schmidt-Chanasit 2,9 , César Muñoz Fontela 2,8 and Beatriz Escudero-Pérez 2,8,* 1 Morcegos de Galicia, Magdalena G-2, 2o izq, 15320 As Pontes de García Rodríguez (A Coruña), Spain; [email protected] 2 WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany; [email protected] (D.C.); [email protected] (A.B.); [email protected] (H.B.); [email protected] (H.H.); [email protected] (A.B.); [email protected] (E.V.N.); [email protected] (S.G.); [email protected] (J.S.-C.); [email protected] (C.M.F.) 3 Laboratoire des Fièvres Hémorragiques en Guinée, Université Gamal Abdel Nasser de Conakry, Commune de Matoto, Conakry, Guinea; [email protected] (F.R.K.); [email protected] (N.M.) 4 Estación Biológica de Doñana, CSIC, 41092 Seville, Spain; [email protected] (J.J.); [email protected] (J.L.G.-M.) 5 CIBER Epidemiology and Public Health (CIBERESP), 28029 Madrid, Spain 6 Public Health England, Porton Down, Wiltshire SP4 0JG, UK; [email protected] 7 Wellcome Centre for Human Genetics, Nuffield Department of Medicine, Oxford University, Oxford OX3 7BN, UK Citation: Hermida Lorenzo, R.J.; 8 German Centre for Infection Research (DZIF), Partner Site Hamburg-Luebeck-Borstel, Cadar, D.; Koundouno, F.R.; Juste, J.; 38124 Braunschweig, Germany Bialonski, A.; Baum, H.; 9 Faculty of Mathematics, Informatics and Natural Sciences, Universität Hamburg, 20148 Hamburg, Germany García-Mudarra, J.L.; Hakamaki, H.; * Correspondence: [email protected] Bencsik, A.; Nelson, E.V.; et al. † These authors contributed equally to this work. Metagenomic Snapshots of Viral Components in Guinean Bats. Abstract: To prevent the emergence of zoonotic infectious diseases and reduce their epidemic Microorganisms 2021, 9, 599. potential, we need to understand their origins in nature. Bats in the order Chiroptera are widely https://doi.org/10.3390/ distributed worldwide and are natural reservoirs of prominent zoonotic viruses, including Nipah microorganisms9030599 virus, Marburg virus, and possibly SARS-CoV-2. In this study, we applied unbiased metagenomic and metatranscriptomic approaches to decipher the virosphere of frugivorous and insectivorous Academic Editor: bat species captured in Guéckédou, Guinea, the epicenter of the West African Ebola virus disease Anne Balkema-Buschmann epidemic in 2013–2016. Our study provides a snapshot of the viral diversity present in these bat species, with several novel viruses reported for the first time in bats, as well as some bat viruses closely Received: 18 February 2021 Accepted: 8 March 2021 related to known human or animal pathogens. In addition, analysis of Mops condylurus genomic DNA Published: 15 March 2021 samples revealed the presence of an Ebola virus nucleoprotein (NP)-derived pseudogene inserted in its genome. These findings provide insight into the evolutionary traits of several virus families in Publisher’s Note: MDPI stays neutral bats and add evidence that nonretroviral integrated RNA viruses (NIRVs) derived from filoviruses with regard to jurisdictional claims in may be common in bat genomes. published maps and institutional affil- iations. Keywords: bats; host; zoonosis; Ebola virus; nonretroviral integrated RNA viruses (NIRVs) Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. Due to their biodiversity, rainforest areas of Central and Western Africa are considered This article is an open access article hotspots for the emergence of zoonotic viruses, and a number of prominent viruses with distributed under the terms and epidemic potential have been identified in this region [1]. Approximately 75% of emerging conditions of the Creative Commons infectious diseases in humans are zoonoses [2,3]. The rate of detection of zoonotic viruses Attribution (CC BY) license (https:// has increased in past decades, possibly due to improved diagnostic capacity and surveil- creativecommons.org/licenses/by/ lance efforts [4,5]. Many novel pathogens that have caused epidemics and pandemics have 4.0/). Microorganisms 2021, 9, 599. https://doi.org/10.3390/microorganisms9030599 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 599 2 of 13 emerged from bats, including Nipah virus, MERS-coronavirus, Marburg virus, and likely Ebola virus (EBOV) and SARS-CoV-2 [6]. Due to the importance of bats as virus reservoirs, it is paramount to regularly investi- gate the bat virome and to assess the potential human pathogenicity of viruses circulating in bats. In this regard, metagenomic analyses of bat viromes can provide relevant infor- mation about viruses circulating in frugivorous and insectivorous bats living in a specific area. Subsequent phylogenetic analyses can evaluate the proximity of bat viruses to known pathogenic human viruses, which may help gauge potential spillover events into humans. For instance, potential novel variants of paramyxovirus and coronaviruses have been shown to commonly circulate in bats [7,8]. In addition, metagenomic analyses of bat viromes have served, for example, to identify novel filoviruses such as Bombali virus in Mops condylurus [9] and Mengla dianlovirus in Rousettus bats [10–13], which provides evidence for bats as reservoirs for Ebola virus (EBOV). Furthermore, EBOV RNA has been detected in three fruit bat species: Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata [14]. Anti-EBOV antibodies have been shown in those species, as well as in Eidolon helvum, Epomophorus gambianus, Micropteropus pusillus, Mops condylurus, Rousettus aegyptiacus, and Rousettus leschenaultii [15]. Finally, in silico analyses of mammalian genomes in the order Mononegavirales have identified nonretroviral sequences derived from single-strand RNA viruses (NIRVs) that are integrated into the genomes of several mammalian species, including bats. Of these, bornavirus NIRVs are the best-characterized [16,17]. Filovirus-derived pseudogenes have also been identified in the genome of bats, marsupials, and rodents [18]. These NIRVs are thought to have their origin in nonhomologous recombination events with genomic transposons during infection [19–21]. Because phylogenetic studies show that these sequences are essentially paleovirus sequences, these findings indicate that filoviruses are ancient and have a long relationship with these mammalian species. In this study, bats captured in the rainforest area of Guéckédou in the Republic of Guinea were sampled via metagenomic and metatranscriptomic studies to characterize the virome of these bat species. In addition, genomic DNA samples were also screened for the presence of possible filovirus-derived nonretroviral integrated RNA virus (NIRV) sequences. The overall goal was to gain insight into the viruses circulating in bats in the area where the 2013–2016 Ebola virus disease (EVD) epidemic started and to underscore the importance of bat surveillance to prevent potential zoonotic outbreaks. 2. Materials and Methods 2.1. Bat Capturing and Sampling The objectives of the present study were communicated to the local community leaders in the Guéckédou prefecture, as well as to the regional government. This study was approved the 7 February 2017 by the Ministère de l’Elèvage et des Productions Animals and the Direction Nationale des Services Vétérinaires de la Republique de Guinée under permit number 015/MEPA/DNSV/2016. The capture and handling of animals and samples were conducted only by trained individuals. Personal safety equipment for capture included leather gloves, goggles, and masks. Bats were sampled in eight locations in the Guéckédou prefecture over the course of five days, between 10 and 15 April 2017. Bats were captured with 9 m and 12 m mist nets at different heights in Guéckédou (Kimberlite garden), Tékoulo (Tékoulo village and Bakama Lela cave), Temessadou (Mongo forest), Nongoa (Nongoa village and Tongdou cave), and Koundou-Lengobengou (Koundou village and Koundou forest) (Figure1). Different environments were sampled: two caves, two construction sites, two secondary forest zones, and two rural core environments. A total of 82 bats were captured by mist nets and kept in cloth bags until sample collection. The forearm length and weight of each specimen were measured. Bats were released after the authors took a blood sample (for virus detection) and a patagium sample (for species identification); these were conserved in AVL and 70% ethanol, respectively. One specimen with a broken wing was euthanized under isoflurane anesthesia and cervical dislocation. Thirteen bats were Microorganisms 2021, 9, 599 3 of 13 found dead due to stress. For these 14 specimens, necropsies were performed to obtain spleen, liver, kidney, and thymus samples, which were preserved in 500 µL of RNAlater. Samples were stored initially at −20 ◦C and later at −80 ◦C. Necropsies were carried out wearing an all-over bodysuit (Tyvek), FFP3 safety mask, face shield, arm protection, and doubled gloves. All other nondisposable equipment was disinfected with 90% ethanol. Nets were dried and disinfected every morning. Bat carcasses were burned after sampling.